Observation of a micro organic specimen including a biological specimen such as bacteria or virus or a biological tissue which has been cut out from a part of a living thing requires the use of a scanning electron microscope. However, with a scanning electron microscope, there are several problems in observing an organic specimen in that an electron beam cannot be applied to a specimen which has not been maintained in vacuum of a predetermined level or higher and the specimen is likely to be damaged by the applied electron beam. In order to solve these problems, an observation method of fixing an organic specimen using paraformaldehyde or the like and coating the surface of the specimen with a conductive material such as gold, platinum, or carbon to protect the surface, an observation method of dyeing a specimen with a heavy metal, and the like have been employed. On the other hand, an observation method for acquiring a high-contrast image of an organic specimen using an electron beam without performing pretreatment on the specimen has been proposed.
For example, Patent Literature 1 discloses a method of observing an internal structure of an organic specimen by detecting a spatial distribution of electrons (tunneling electrons) passing through the organic specimen in a scanning electron microscope. An organic specimen is attached to a conductive film of a stacked body of an insulating film and a conductive film and the insulating film is irradiated with an electron beam from an electron gun. At this time, secondary electrons generated in the insulating film form a large potential gradient in the stacked body and are discharged to the conductive film due to a tunneling effect. These electrons also pass through the organic specimen by tunneling and an image reflecting the internal structure of the organic specimen is obtained by detecting the passing electrons using a secondary electron detector. Since an organic specimen is not directly irradiated with an electron beam from an electron gun and thus damage to the specimen is small, the pretreatment on the organic specimen can be skipped.
Non Patent Literature 1 describes a method of observing an organic specimen in a scanning electron microscope with the organic specimen immersed in an aqueous solution. When a metal film is formed on an insulating film and is then irradiated with an electron beam, local potential change is caused and an attenuation state when the electron beam passes through the organic specimen in the aqueous solution can be observed as an image (a changing potential transmission observation method). Such a method employs the following phenomenon. That is, since the specific dielectric constant of water is about 80 which is large, water is not affected by the potential change and transmits the electron beam. Since the specific dielectric constant of an organic specimen ranges from about 2 to 3 which is small, the organic specimen is greatly affected by the potential change and attenuates the electron beam. Because an organic specimen is not directly irradiated with an electron beam from an electron gun, the organic specimen does not have to be disposed in a vacuum, and pretreatment on the organic specimen can be skipped.
Similarly, Patent Literature 2 discloses a method of observing an organic specimen which is maintained in an aqueous solution in a scanning electron microscope. Specifically, an organic specimen is interposed with an aqueous solution between a pair of insulating films opposing each other, a conductive film formed on an outward surface of one insulating film is scanned and irradiated with an electron beam while the intensity thereof is changed in a pulse shape, and potential change on an outward surface of the other insulating film is detected. When a part of the conductive film on which an electron beam is incident has a negative charge, electric dipoles of water molecules in the aqueous solution are arranged in accordance with a potential gradient, but this state is released by blocking the electron beam. When ON/OFF of an electron beam is repeated at a frequency of 1 kHz or higher and signals of the same frequency components are extracted by the detection side, potential change can be separated with a high resolution. By narrowing an irradiation diameter of an electron beam to about 1 nm, a resolution of 1 nm at the same level is obtained.